Nanoinjection

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Nanoinjection is the process of using a microscopic lance and electrical forces to deliver DNA to a cell. It is claimed to be more effective than microinjection because the lance used is ten times smaller than a micropipette and the method uses no fluid. The nanoinjector mechanism is operated while submerged in a pH buffered solution. Then, a positive electrical charge is applied to the lance, which accumulates negatively charged DNA on its surface. The nanoinjector mechanism then penetrates the zygotic membranes, and a negative charge is applied to the lance, releasing the accumulated DNA within the cell. The lance is required to maintain a constant elevation on both entry and exit of the cell. [1]

Contents

Nanoinjection results in a long-term cell viability of 92% following the electrophoretic injection process with a 100 nm diameter nanopipette, the typical diameter of nanoinjection pipet. [2]

Single cell transfections are used to virtually transfer any type of mammalian cell into another using a syringe which creates an entry for DNA to be released. A nano needle is used as a mechanical vector for plasmid DNA. This is called Atomic Force Microscopy or AFM. The purpose is to not cause permanent damage to the cell or provoke cellular leaking of intracellular fluid. AFM is a tool of choice as it allows for precise positioning of the DNA. This is important because it allows for tip penetration into the cytosol, which is critical for viable DNA transfer into the cell. [3]

Reasons to use nanoinjection include the insertion of genetic material into the genome of a zygote. This method is a critical step in understanding and developing gene functions.

Nanoinjection is also used to genetically modify animals to aid in the research of cancer, Alzheimer’s disease, and diabetes. [2]

Fabrication

The lance is made using the polyMUMPs fabrication technology.  It creates a gold layer, and two structural layers that are 2.0 and 1.5 μm thick respectively.  It is a simple process, which makes it good as a platform to prototype polysilicon MEMS devices at a low commercial cost of fabrication.  The lance has a solid, tapered body, that is 2 μm thick, with a tip width of 150 nm.  The taper is set at 7.9°, coming to a maximum width of 11 μm. Two highly folded electrical connections provide an electrical path between the lance and two equivalent bond pads, with a gold wire connecting one of the bond pads to an integrated circuit chip carrier’s pin.  The carrier is then placed into a custom built electrical socket. [4]

In the situation of fertilizing eggs, the lance is incorporated into a kinematic mechanism consisting of a change-point parallel-guiding six-bar mechanism and a compliant parallel-guiding folded-beam suspension.

Techniques

Electrophoretic Injection

Electrophoretic injection remains the most common form of nanoinjection. Just as with the other methods, a lance ten times smaller than that of microinjection is used. Preparing the lance for injection, a positive charge is applied, attracting the negatively-charged DNA to its tip. After the lance has reached a desired depth within the cell, the charge is reversed, repelling the DNA into the cell. [1] The typical injection voltages are ±20 V, but can be as low as 50-100 mV.

Diffusion

A manual force is applied to a center fixture of the injection device, moving the lances through cell membranes and into the cytoplasm or nucleus of adhered cells. The magnitude of the force is measured using a force plate on a small number of injections to obtain an estimate of the manual force. The force plate is arranged to measure the force actually applied to the injection chip (that is, not including the stiffness of the support spring). After holding the force for five seconds, the force is released and the injection device is removed from the cell. The diffusion protocol presented data for comparison against other variations in the injection process. [5]

Applications

By delivering certain particles into cells, diseases can be treated or even cured. Gene therapy is possibly the most common field of foreign material delivery into cells and has great implications for curing human genetic diseases.

For example, two monkeys colorblind from birth were given gene therapy treatment in a recent experiment. As a result of gene therapy, both animals had their color vision restored with no apparent side effects. Traditionally, gene therapy has been divided into two categories: biological (viral) vectors and chemical or physical (nonviral) approaches. Although viral vectors are currently the most effective approach to delivering DNA into cells, they have certain limitations, including immunogenicity, toxicity, and limited capacity to carry DNA. [5]

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, an ideal vector system has not yet been constructed. [6]

Alternatives

Microinjection is the predecessor to nanoinjection. Still used in biological research, microinjection is useful in the examination of non-living cells or in cases where cell viability does not matter. Using a glass pipette 0.5-1.0 micrometers in diameter, the cell has its membrane damaged upon puncture. As opposed to nanoinjection, microinjection uses DNA-filled liquid driven into the cell under pressure. Depending on factors such as the skill of the operator, survival rates of cells undergoing this procedure can be as high as 56% or as low as 9%. [2]

Other methods exist that target groups of cells, such as electroporation. These methods are incapable of targeting specific cells, and are therefore not usable where efficiency and cell viability are a concern.

Related Research Articles

<span class="mw-page-title-main">Electroporation</span> Method in molecular biology to introduce DNA into other hosts

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introduced into the cell. In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred, cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across suspended cells in an electroporation cuvette. Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective in increasing cell membrane's permeability than chemical transformation.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

In the field of genetics, a suicide gene is a gene that will cause a cell to kill itself through the process of apoptosis. Activation of a suicide gene can cause death through a variety of pathways, but one important cellular "switch" to induce apoptosis is the p53 protein. Stimulation or introduction of suicide genes is a potential way of treating cancer or other proliferative diseases.

<span class="mw-page-title-main">Transduction (genetics)</span> Transfer process in genetics

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

<span class="mw-page-title-main">Microinjection</span>

Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x.

Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture. Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes or other genetic material by a vector is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage λ to infect monkey kidney cells maintained in culture.

Impalefection is a method of gene delivery using nanomaterials, such as carbon nanofibers, carbon nanotubes, nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. Plasmid DNA containing the gene, and intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s).

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection systems, and viruses. This method offers advantages such as high transfection efficiency and biocompatibility which are balanced with limitations.

Nucleofection is an electroporation-based transfection method which enables transfer of nucleic acids such as DNA and RNA into cells by applying a specific voltage and reagents. Nucleofection, also referred to as nucleofector technology, was invented by the biotechnology company Amaxa. "Nucleofector" and "nucleofection" are trademarks owned by Lonza Cologne AG, part of the Lonza Group.

<span class="mw-page-title-main">Sonoporation</span> Technique in molecular biology

Sonoporation, or cellular sonication, is the use of sound in the ultrasonic range for increasing the permeability of the cell plasma membrane. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation. Sonoporation employs the acoustic cavitation of microbubbles to enhance delivery of these large molecules. The exact mechanism of sonoporation-mediated membrane translocation remains unclear, with a few different hypotheses currently being explored.

Magnet-assisted transfection is a transfection method which uses magnetic interactions to deliver DNA into target cells. Nucleic acids are associated with magnetic nanoparticles, and magnetic fields drive the nucleic acid-particle complexes into target cells, where the nucleic acids are released.

Optical transfection is a biomedical technique that entails introducing nucleic acids into cells using light. All cells are surrounded by a plasma membrane, which prevents many substances from entering or exiting the cell. Lasers can be used to burn a tiny hole in this membrane, allowing substances to enter. This is tremendously useful to biologists who are studying disease, as a common experimental requirement is to put things into cells.

<span class="mw-page-title-main">Vectors in gene therapy</span>

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.

Gene therapy is being studied as a treatment for osteoarthritis (OA). Unlike pharmacological treatments which are administered systemically, gene therapy aims to establish sustained, synthesis of gene products and tissue rehabilitation within the joint.

Tissue nanotransfection (TNT) is an electroporation-based technique capable of gene and drug cargo delivery or transfection at the nanoscale. Furthermore, TNT is a scaffold-less tissue engineering (TE) technique that can be considered cell-only or tissue inducing depending on cellular or tissue level applications. The transfection method makes use of nanochannels to deliver cargo to tissues topically. 

<span class="mw-page-title-main">Intracellular delivery</span> Scientific research area

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.

<span class="mw-page-title-main">Hydrodynamic delivery</span> Gene Transfer Method

Hydrodynamic Delivery (HD) is a method of DNA insertion in rodent models. Genes are delivered via injection into the bloodstream of the animal, and are expressed in the liver. This protocol is helpful to determine gene function, regulate gene expression, and develop pharmaceuticals in vivo.

References

  1. 1 2 Aten, Quentin T.; Jensen, Brian D.; Burnett, Sandra H.; Howell, Larry L. (2014). "A self-reconfiguring metamorphic nanoinjector for injection into mouse zygotes". Review of Scientific Instruments. 85 (5): 055005. Bibcode:2014RScI...85e5005A. doi:10.1063/1.4872077. PMID   24880406.
  2. 1 2 3 Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon (2017-01-25). "Survival rate of eukaryotic cells following electrophoretic nanoinjection". Scientific Reports. 7: 41277. Bibcode:2017NatSR...741277S. doi:10.1038/srep41277. ISSN   2045-2322. PMC   5264641 . PMID   28120926.
  3. ↑ Cuerrier, Charles M.; Lebel, Réjean; Grandbois, Michel (2007-04-13). "Single cell transfection using plasmid decorated AFM probes". Biochemical and Biophysical Research Communications. 355 (3): 632–636. doi:10.1016/j.bbrc.2007.01.190. ISSN   0006-291X. PMID   17316557.
  4. ↑ Aten, Q. T.; Jensen, B. D.; Burnett, S. H.; Howell, L. L. (December 2011). "Electrostatic Accumulation and Release of DNA Using a Micromachined Lance". Journal of Microelectromechanical Systems. 20 (6): 1449–1461. doi:10.1109/JMEMS.2011.2167658. ISSN   1057-7157. S2CID   59961.
  5. 1 2 Lindstrom, Zachary K.; Brewer, Steven J.; Ferguson, Melanie A.; Burnett, Sandra H.; Jensen, Brian D. (2014-10-03). "Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array". Journal of Nanotechnology in Engineering and Medicine. 5 (2): 021008–021008–7. doi:10.1115/1.4028603. ISSN   1949-2944. S2CID   135872805.
  6. ↑ Mehierhumbert, S.; Guy, R. (2005-04-05). "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells". Advanced Drug Delivery Reviews. 57 (5): 733–753. doi:10.1016/j.addr.2004.12.007. ISSN   0169-409X. PMID   15757758.
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